Regulation of carbohydrate and fatty acid utilization by L-carnitine during cardiac development and hypoxia

This study is designed to investigate whether substrate preference in the myocardium during the neonatal period and hypoxia-induced stress is controlled intracellularly or by extracellular substrate availability. To determine this, the effect of exogenous L-carnitine on the regulation of carbohydrate and fatty acid metabolism was determined during cardiac stress (hypoxia) and during the postnatal period. The effect of L-carnitine on long chain (palmitate) and medium chain (octonoate) fatty acid oxidation was studied in cardiac myocytes isolated from less than 24 h old (new born; NB), 2 week old (2 week) and hypoxic 4 week old (HY) piglets. Palmitate oxidation was severely decreased in NB cells compared to those from 2 week animals (0.456 ± 0.04 vs. 1.207 ± 0.52 nmol/mg protein/30 min); surprisingly, cells from even older hypoxic animals appeared shifted toward the new born state (0.695 ± 0.038 nmol/mg protein/30 min). Addition of L-carnitine to the incubation medium, which stimulates carnitine palmitoyl-transferase I (CPTI) accelerated palmitate oxidation 3 fold in NB and approximately 2 fold in HY and 2 week cells. In contrast, octanoate oxidation which was greater in new born myocytes than in 2 week cells, was decreased by L-carnitine suggesting a compensatory response. Furthermore, oxidation of carbohydrates (glucose, pyruvate, and lactate) was greatly increased in new born myocytes compared to 2 week and HY cells and was accompanied by a parallel increase in pyruvate dehydrogenase (PDH) activity. The concentration of malonyl-CoA, a potent inhibitor of CPTI was significantly higher in new born heart than at 2 weeks. These metabolic data taken together suggest that intracellular metabolic signals interact to shift from carbohydrate to fatty acid utilization during development of the myocardium. The decreased oxidation of palmitate in NB hearts probably reflects decreased intracellular L-carnitine and increased malonyl-CoA concentrations. Interestingly, these data further suggest that the cells remain compliant so that under stressful conditions, such as hypoxia, they can revert toward the neonatal state of increased glucose utilization.     

A monoclonal antibody targeting a highly conserved epitope in influenza B neuraminidase provides protection against drug resistant strains

All influenza viral neuraminidases (NA) of both type A and B viruses have only one universally conserved sequence located between amino acids 222–230. A monoclonal antibody against this region has been previously reported to provide broad inhibition against all nine subtypes of influenza A NA; yet its inhibitory effect against influenza B viral NA remained unknown. Here, we report that the monoclonal antibody provides a broad inhibition against various strains of influenza B viruses of both Victoria and Yamagata genetic lineage. Moreover, the growth and NA enzymatic activity of two drug resistant influenza B strains (E117D and D197E) are also inhibited by the antibody even though these two mutations are conformationally proximal to the universal epitope. Collectively, these data suggest that this unique, highly-conserved linear sequence in viral NA is exposed sufficiently to allow access by inhibitory antibody during the course of infection; it could represent a potential target for antiviral agents and vaccine-induced immune responses against diverse strains of type B influenza virus.     

Aggregate Clearance of α-Synuclein in Saccharomyces cerevisiae Depends More on Autophagosome and Vacuole Function Than on the Proteasome

Parkinson disease is the second most common neurodegenerative disease. The molecular hallmark is the accumulation of proteinaceous inclusions termed Lewy bodies containing misfolded and aggregated α-synuclein. The molecular mechanism of clearance of α-synuclein aggregates was addressed using the bakers' yeast Saccharomyces cerevisiae as the model. Overexpression of wild type α-synuclein or the genetic variant A53T integrated into one genomic locus resulted in a gene copy-dependent manner in cytoplasmic proteinaceous inclusions reminiscent of the pathogenesis of the disease. In contrast, overexpression of the genetic variant A30P resulted only in transient aggregation, whereas the designer mutant A30P/A36P/A76P neither caused aggregation nor impaired yeast growth. The α-synuclein accumulation can be cleared after promoter shut-off by a combination of autophagy and vacuolar protein degradation. Whereas the proteasomal inhibitor MG-132 did not significantly inhibit aggregate clearance, treatment with phenylmethylsulfonyl fluoride, an inhibitor of vacuolar proteases, resulted in significant reduction in clearance. Consistently, a cim3-1 yeast mutant restricted in the 19 S proteasome regulatory subunit was unaffected in clearance, whereas an Δatg1 yeast mutant deficient in autophagy showed a delayed aggregate clearance response. A cim3-1Δatg1 double mutant was still able to clear aggregates, suggesting additional cellular mechanisms for α-synuclein clearance. Our data provide insight into the mechanisms yeast cells use for clearing different species of α-synuclein and demonstrate a higher contribution of the autophagy/vacuole than the proteasome system. This contributes to the understanding of how cells can cope with toxic and/or aggregated proteins and may ultimately enable the development of novel strategies for therapeutic intervention.     

Relationships between the Circadian System and Alzheimer's Disease-Like Symptoms in Drosophila

Circadian clocks coordinate physiological, neurological, and behavioral functions into circa 24 hour rhythms, and the molecular mechanisms underlying circadian clock oscillations are conserved from Drosophila to humans. Clock oscillations and clock-controlled rhythms are known to dampen during aging; additionally, genetic or environmental clock disruption leads to accelerated aging and increased susceptibility to age-related pathologies. Neurodegenerative diseases, such as Alzheimer''s disease (AD), are associated with a decay of circadian rhythms, but it is not clear whether circadian disruption accelerates neuronal and motor decline associated with these diseases. To address this question, we utilized transgenic Drosophila expressing various Amyloid-β (Aβ) peptides, which are prone to form aggregates characteristic of AD pathology in humans. We compared development of AD-like symptoms in adult flies expressing Aβ peptides in the wild type background and in flies with clocks disrupted via a null mutation in the clock gene period (per⁰¹). No significant differences were observed in longevity, climbing ability and brain neurodegeneration levels between control and clock-deficient flies, suggesting that loss of clock function does not exacerbate pathogenicity caused by human-derived Aβ peptides in flies. However, AD-like pathologies affected the circadian system in aging flies. We report that rest/activity rhythms were impaired in an age-dependent manner. Flies expressing the highly pathogenic arctic Aβ peptide showed a dramatic degradation of these rhythms in tune with their reduced longevity and impaired climbing ability. At the same time, the central pacemaker remained intact in these flies providing evidence that expression of Aβ peptides causes rhythm degradation downstream from the central clock mechanism.     

The naphthoquinol oxidizing cytochrome bc1 complex of the hyperthermophilic knallgasbacterium Aquifex aeolicus: properties and phylogenetic relationships

Phylogenetic analysis of constituent proteins of Rieske/cytochrome b complexes [Schütz et al. (2000) J. Mol. Biol. 300, 663-675] indicated that the respective enzyme from the hyperthermophile Aquifex (A.) aeolicus is closely related to proteobacterial counterparts, in disagreement with positioning of its parent species on small subunit rRNA trees. An assessment of the details and possible reasons for this discrepancy necessitates a thorough understanding of the biochemical and biophysical properties of the enzyme in addition to the bioinformatic data. The cytochrome bc(1) complex from A. aeolicus, which is part of the "Knallgasreaction" pathway, was therefore studied in membranes and in detergent-solubilized, isolated complex. Hemes b(L) (E(m,7) = -190 mV; g(z)= 3.7), b(H) (E(m,7) = -60 mV; g(z )= 3.45), and c(1) (E(m,7) = +160 mV; g(z )= 3.55) were identified by EPR and optical spectroscopy in combination with electrochemical methods. Two electrochemically distinct (E(m,7) = +95 mV; E(m,7) = +210 mV) Rieske centers were detected in membranes, and the +210 mV species was shown to correspond to the Rieske center of the cyt bc(1) complex. The gene coding for this latter Rieske protein was heterologously expressed in Escherichia coli, and the resulting protein was characterized in detail. The pool quinone of A. aeolicus was determined to be naphthoquinone. The redox poises of the individual electron-transfer steps are compared to those of other Rieske/cyt b complexes. The Aquifex enzyme was found to represent the only extant naphthoquinol oxidizing true cyt bc(1) complex described so far. An improved scenario for the phylogenetic positioning of the Aquifex cyt bc(1) complex is proposed.     

A microdeletion of less than 250 kb, including the proximal part of the FMR-1 gene and the fragile-X site, in a male with the clinical phenotype of fragile-X syndrome

A gene designated "FMR-1" has been isolated at the fragile-X locus. One exon of this gene is carried on a 5.1-kb EcoRI fragment that exhibits length variation in fragile-X patients because of amplification of or insertion into a CGG-repeat sequence. This repeat probably represents the fragile site. The EcoRI fragment also includes an HTF island that is hypermethylated in fragile-X patients showing absence of FMR-1 mRNA. In this paper, we present further evidence that the FMR-1 gene is involved in the clinical manifestation of the fragile-X syndrome and also in the expression of the cellular phenotype. A deletion including the HTF island and exons of the FMR-1 gene was detected in a fragile X-negative mentally retarded male who presented the clinical phenotype of the fragile-X syndrome. The deletion involves less than 250 kb of genomic DNA, including DXS548 and at least five exons of the FMR-1 gene. These data support the hypothesis that loss of function of the FMR-1 gene leads to the clinical phenotype of the fragile-X syndrome. In the fragile-X syndrome, there are pathogenetic mechanisms other than amplification of the CGG repeat that do have the same phenotypic consequences.     

Nomenclatural adjustments in some European monocotyledons

New nomenclatural combinations are validated for fifteen taxa belonging to the generaDanthonia, Stipa, Lolium, Phippsia, Elymus, Schoenoplectus andAllium.